Research Article
Synthesis and Pharmacological Evaluation of New
Pyridazin-Based Thioderivatives as Formyl Peptide
Receptor (FPR) Agonists
Letizia Crocetti,1Claudia Vergelli,1Agostino Cilibrizzi,1Alessia Graziano,1
Andrei I. Khlebnikov,2Liliya N. Kirpotina,3Igor A. Schepetkin,3Mark T. Quinn,3and
Maria Paola Giovannoni1*
1Dipartimento di Scienze Farmaceutiche, Sesto Fiorentino 50019, Firenze, Italy 2Department of Chemistry, Altai State Technical University, Barnaul, Russia 3Department of Immunology and Infectious Diseases, Montana State University, Bozeman,
Montana 59717, USA
Strategy, Management and Health Policy Enabling Technology, Genomics, Proteomics Preclinical Research Preclinical Development Toxicology, Formulation Drug Delivery, Pharmacokinetics Clinical Development Phases I-III Regulatory, Quality, Manufacturing Postmarketing Phase IV
ABSTRACT A new series of pyridazinone-based thioderivatives and pyridazine analogs was synthe-sized and tested for their ability to bind to the three human formyl peptide receptor (FPR) isoforms (FPR1, FPR2, and FPR3) and to activate intracellular calcium mobilization and chemotaxis in human neutrophils. Among the pyridazin-3(2H)-one derivatives tested, analogs 8b and 8c were mixed FPR1/FPR2 agonists, with median effective concentration values in the micromolar range, and were able to activate chemotaxis and Ca2+flux in human neutrophils in the low micromolar range. Molecular docking studies showed that interaction of a ligand with Arg205 of FPR1 is important for FPR1 agonist activity. For FPR2, differences in activity between oxygen-containing compounds and their thio-analogs were due to steric bulkiness of sulfur-containing groups. Drug Dev Res 74 : 259–271, 2013. ©2013 Wiley Periodicals, Inc.
Key words: pyridazine; pyridazin-3(2H)-one; formyl peptide receptors; neutrophil; chemotaxis; Ca2+mobilization
INTRODUCTION
Formyl peptide receptors (FPRs) are a family of chemoattractant receptors that play an essential role in host defense against infection and trauma. In addition, they are involved at different levels in the regulation of inflammatory reactions and sensing cellular dysfunction [Migeotte et al., 2006]. FPRs belong to the seven trans-membrane domain G-protein-coupled receptor family, are expressed in the majority of white blood cells, and are important in host defense and inflammation [Ye et al., 2009]. All major neutrophil functions accom-plished through FPRs can be inhibited by treatment with pertussis toxin [Bokoch and Gilman, 1984], indi-cating that the G proteins coupled to FPRs belong to the Gi family of heterotrimeric proteins [Simon
et al., 1991]. Activation of FPRs induces a variety of
responses, e.g. directional movement of neutrophils, lysosomal enzyme release [Schiffmann et al., 1975], degranulation, and production of superoxide anion [Prossnitz and Ye, 1997; Mills et al., 1999; Le et al., 2002]. Three FPR subtypes have been identified in humans (FPR1, FPR2, and FPR3) [Ye et al., 2009]. FPR1 was the first that was biochemically defined and was a high-affinity binding site on the surface of *Correspondence to: Maria Paola Giovannoni, Diparti-mento di Scienze Farmaceutiche, Via Ugo Schiff 6, Sesto Fiorentino 50019, Firenze, Italy.
E-mail: [email protected]
Received 23 January 2013; Accepted 4 February 2013 Published online in Wiley Online Library (wileyonlinelibrary. com). DOI: 10.1002/ddr.21076
neutrophils for the prototypic peptide formyl-methionine-leucine-phenylalanine (fMLF) [Ye et al., 2009]. FPR2 shares 69% amino acid identity with human FPR1, but despite the relatively high level of sequence homology, is a low-affinity receptor for fMLF [Quehenberger et al., 1993]. FPR3 shares 56% and 83% sequence identity with human FPR1 and FPR2, respectively, but does not bind N-formyl peptides, e.g. fMLF. Instead, FPR3 responds to some nonformylated chemotactic peptides identified as FPR2 agonists.
FPRs interact with a wide range of structurally different pro- and anti-inflammatory ligands associated with different diseases, including amyloidosis and Alzheimer’s disease [Cui et al., 2002], some types of cancers and related alopecia induced by most antican-cer agents [Edwards et al., 2005; Tsuruky et al., 2007], prion diseases [Zhou et al., 2009], HIV [Kilby et al., 1998], stomach ulcers [De Paulis et al., 2004], and nociception associated with inflammatory processes [Pieretti et al., 2004]. Many studies indicate that FPRs are important mediators of key events in endogenous anti-inflammation, and it has been proposed that FPR modulation is an attractive approach for the targeting biochemical processes for resolving inflammation [Gilroy et al., 2004]. Likewise, FPRs may represent therapeutic targets for selectively stimulating innate immune responses [Serhan et al., 2007; Zhang and Falla, 2009; Dufton and Perretti, 2010].
Previous studies led to the identification of a wide range of structurally unrelated nonpeptide and peptide FPR agonists, including synthetic molecules and both host-derived and pathogen-derived agents [Bokoch et al., 1984; Gierschik et al., 1989], and more recently, we identified a large number of potent FPR agonists with a pyridazin-3(2H)-one scaffold that contained a meth-oxybenzyl group at position 4 [Cilibrizzi et al., 2009, 2012]. Two of the most interesting compounds of the series are shown in Figure 1: compound 1 is a mixed FPR1/FPR2 agonist with activity in the low micromolar range (median effective concentration values [EC50] =
3.4 and 3.8 mM for FPR1 and FPR2, respectively), whereas compound 2 is a FPR2-specific agonist (EC50=
2.4 mM). Structural analysis of this series of compounds indicated that an acetamide spacer at N-2 of the pyridazi-none ring is an essential requirement for specificity and potency of these compounds, and the role of both C = O (H-bond acceptor) and amidic NH (H-bond donor) in the side chain was crucial for binding to FPRs. More-over, the presence of a lipophilic and/or electronegative
para substituent in the aromatic system of the side chain
at position 2 was an important element required for potency and selectivity [Cilibrizzi et al., 2009].
Based on previous reports demonstrating benefi-cial substitutions of C = S for C = O and methylthio (SCH3) for methoxy (OCH3) [Wrobel et al., 1989;
Güngör et al., 2006], we designed a series of thio-analogs characterized by properties similar to those of previously reported parent compounds [Cilibrizzi et al., 2009]. Furthermore, a series of pyridazine analogs where the phenylacetamide moiety was moved to position 3 of the ring was also designed. For all new compounds, we report herein the synthesis and biological evaluation for FPR agonist activity, selectivity versus FPR1/FPR2/ FPR3, and the ability to activate intracellular calcium mobilization and chemotaxis in human neutrophils.
MATERIALS AND METHODS
Chemistry
Syntheses of the new pyridazin-3(2H)-one and pyridazine thioderivatives are depicted in Figures 2 and 3. In Figure 2, procedures to obtain the pyridazinone-3(2H)-one thioderivatives 8a-c, 9 and the thiopyridazine analog 11 are reported. In both cases, the starting material was the 6-methyl-4,5-dihydropyridazin-3(2H)-one compound 4 [Meng and Hesse, 1990], which was directly converted in the new benzyl derivative 5a and the previously described 5b,c [Cilibrizzi et al., 2009] by Knoevenagel condensa-tion using the appropriate aromatic aldehyde in the
N N O O H N Br N N O O H N Br O 2 2.4 μM (FPR2) O 1 3.4 μM (FPR1) 3.8 μM (FPR2) EC50= EC50= N N O O H N O O 3 7.7 μM (FPR1) 14.4 μM (FPR2) EC50=
presence of KOH in absolute EtOH (5% w/v). Alkyla-tion of intermediates 5a, 5b with ethyl bromoacetate resulted in the esters 6a, 6b, [Cilibrizzi et al., 2009], which after alkaline hydrolysis gave the corresponding acids 7a,b [Cilibrizzi et al., 2009]. Subsequent reaction of 7a,b with ethyl chloroformate in THF in presence of triethylamine resulted in the intermediate mixed anhy-drides, which in turn were transformed in good yields into the final amide 1 [Cilibrizzi et al., 2009] and the new compounds 8a-c. Moreover, the thioamide analog 9 was directly generated from compound 1 by treat-ment with Lawesson’s reagent in toluene at 80°C [Jesberger et al., 2003]. Considering that compound 1 has two carbonyl groups susceptible to thionation, both
1H NMR and MS(ESI) analyses were performed to
confirm structure 9. MS analysis clearly demonstrated the presence of a fragment of compound 9 containing the pyridazinone nucleous, and1H NMR experiments
showed a clear difference in the chemical shift of the signal related to N-CH2-CO in 1 and the corresponding
N-CH2-CS in compound 9. These findings suggest that
the pyridazinone ring was unchanged, and the sulfur
was present only and univocally on the side chain. Figure 2 also depicts the synthetic procedure for obtaining the pyridazine analog 11. Using Lawesson’s reagent, as described above, the precursor 5c [Cilibrizzi et al., 2009] was transformed into the intermediate 10 which, in turn, was alkylated under standard conditions with N-(4-bromophenyl)-2-chloroacetamide [Baraldi et al., 2007] to obtain the final compound 11, contain-ing the phenylacetamide moiety at C-3 of the rcontain-ing.
The synthetic procedure to obtain pyridazine analogs 15a,b and 18 is depicted in Figure 3. The previously described 3-chloro-6-phenylpyridazin-4-ol, compound 12 [Sircar, 1983] was converted into intermediate 13 by performing a cross-coupling reac-tion with the appropriate aryl boronic acids, using copper acetate as catalyst and a weak base (triethy-lamine) in CH2Cl2 [Quach and Batey, 2003; Chiang
and Olsson, 2004]. Compound 13 was then treated with saturated ethanolic ammonia at 180°C to obtain the corresponding 3-amino derivative 14, which was directly reacted under standard conditions with the 4-bromophenylisocyanate or the 4-bromobenzoyl
HN N O HN N O R N N O R O O N N O R OH O N N O R NH O R1 N N O NH S Br O HN N S O N N O HS N N O S O NH Br 10 B A 11 9 1, 8a-c 7a,b 6a,b 5a-c 4 for 5c for 1 F E E D C 5 a b c R SCH3 (p) OCH3 (m) OCH3 (p) 6, 7 a b R SCH3 (p) OCH3 (m) comp 1 8a 8b 8c R OCH3 (m) SCH3 (p) SCH3 (p) OCH3 (m) R1 Br Br I SCH3
Fig. 2. Synthesis of pyridazin-3(2H)-one-based thioderivatives 8a-c, 9 and pyridazine analog 11.
Reagents and conditions: (A) substituted benzaldehyde (1 equiv), KOH 5% (w/v) in anhydrous EtOH, 1–5 h, reflux; (B) ethyl bromoacetate (1.5 equiv), K2CO3(2 equiv), anhydrous CH3CN, 2–4 h, reflux; (C) NaOH 6 N, 1–2 h, 80°C; (D) ethyl chloroformate (1.1 equiv), Et3N (3.5 equiv),
substituted aniline (2 equiv), anhydrous THF, 12 h, -5°C → rt; (E) Lawesson’s reagent (2 equiv), anhydrous toluene, 2–3 h, 80°C; (F)
chloride to obtain the urea 15a and the amide 15b, respectively. Alternatively, final compound 18 was pre-pared from intermediate 14 by performing the same reaction sequence (alkylation with ethyl bromoacetate, alkaline hydrolysis, and finally, through the intermedi-ate mixed anhydride, coupling with 4-bromoaniline) shown in Figure 2 for compounds 1 and 8a-c.
Experimental
All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). Extracts were dried over Na2SO4, and the solvents were removed under reduced
pressure. All reactions were monitored by thin layer chromatography (TLC) using commercial plates pre-coated with Merck silica gel 60 F-254 (Merck, Darms-tadt, Germany). Visualization was performed by UV fluorescence (lmax= 254 nm) or by staining with iodine
or potassium permanganate. Chromatographic separa-tions were performed on a silica gel column by gra-vity chromatography (Kieselgel 40, 0.063–0.200 mm; Merck) or flash chromatography (Kieselgel 40, 0.040– 0.063 mm; Merck). All melting points were determined on a microscope hot stage Büchi apparatus (Assago, Milano, Italy) and are uncorrected. Yields refer to chromatographically and spectroscopically pure com-pounds, unless otherwise stated. The identity and purity of intermediates and final compounds were ascertained
through 1H NMR and TLC. 1H NMR spectra were
recorded with Avance 400 instruments (Bruker Biospin Version 002, Bruker AXS Inc., Madison, WI USA with full digital signal generation [SGU]). Chemical shifts (d) were reported in ppm to the nearest 0.01 ppm, using the solvent as the internal standard. Coupling constants (J-values) are given in Hz and were calculated using TopSpin 1.3 software (Bruker) rounded to the nearest 0.1 Hz. Mass spectra (m/z) were recorded on an ESI-MS triple quadrupole mass spectrometer (Varian 1200L, Sunnyvale, CA USA). Microanalyses were per-formed with a Perkin-Elmer 260 elemental analyzer (Waltham, MA USA) for C, H, and N, and the results were within⫾ 0.4% of the theoretical values, unless otherwise stated.
General procedure for compounds (5a-c)
To 12 ml of a solution of KOH in absolute EtOH (5%, w/v), compound 4 (4.46 mmol) and the appropri-ate aromatic aldehyde (4.46 mmol) were added, and the solution was refluxed under stirring for 1–5 h. After cooling, the mixture was concentrated in vacuo, diluted with ice-cold water (20–25 ml) and acidified with 2 N HCl. The suspension was extracted with CH2Cl2
(3 ¥ 25 ml), and removal of the solvent resulted in com-pounds 5a-c, which were purified by crystallization in ethanol. N N Cl HO N N Cl O O N N H2N O O N N HN O O X Br N N HN O O O O N N HN O O O OH N N HN O O O HN F E D C B A 12 18 17 16 15a,b 14 13 15 a b X CONH CO Br
Fig. 3. Synthesis of pyridazine-based analogs 15a,b and 18.
Reagents and conditions: (A) 4-methoxyphenylboronic acid (2 equiv), Cu(OAc)2(1.5 equiv), Et3N (2 equiv), CH2Cl2, 12 h, rt; (B) NH3/anhydrous
EtOH, 7 h, 180°C; (C) for 15a: 4-bromophenylisocyanate (1.1 equiv) anhydrous toluene, 6 h, reflux; for 15b: 4-bromobenzoyl chloride (4 equiv), Et3N (catalytic), anhydrous CH2Cl2, 24 h, 0°C/rt; (D) ethyl bromoacetate (3 equiv), K2CO3(2 equiv), anhydrous CH3CN, 8 h, reflux; (E) NaOH
6-Methyl-4-[4-(methylthio)benzyl]pyridazin-3(2H)-one (5a)
Mp = 151–153°C; crystallization solvent = EtOH; yield = 46%. 1H NMR (CDCl
3), d: 2.26 (3H, s), 2.51
(3H, s), 3.87 (2H, s), 6.71 (1H, s), 7.18 (2H, d,
J = 8.2 Hz), 7.27 (3H, dd, J = 9.3, 1.8 Hz).
General procedure for compounds (6a,b)
A mixture of the suitable intermediate 5a,b (4.50 mmol), K2CO3(9.00 mmol), and ethyl
bromoac-etate (6.75 mmol) in CH3CN (10 ml) was refluxed
under stirring for 2–4 h. The mixture was then concen-trated in vacuo, diluted with cold water, and extracted with CH2Cl2(3 ¥ 15 ml). The solvent was evaporated in
vacuo, and compounds 6a and b were purified by
crys-tallization from ethanol.
Ethyl-2-{3-methyl-5-[4-(methylthio)benzyl]-6-oxopyridazin-1(6H)-yl}acetate (6a)
Mp = 129–131°C; crystallization solvent = EtOH; yield = 99.9%. 1H NMR (CDCl
3), d: 1.31 (3H, t, J =
6.8 Hz), 2.24 (3H, s), 2.51 (3H, s), 3.87 (2H, s), 4.27 (2H, q, J = 6.8 Hz), 4.86 (2H, s), 6.68 (1H, s), 7.17 (2H, d, J = 7.3 Hz), 7.25–7.29 (2H, m).
General procedure for compounds (7a,b)
A suspension of the intermediate 6a and 6b (4.40 mmol) in 6 N NaOH (10 ml) was stirred at 80°C for 1–2 h. The mixture was first diluted with ice-cold water and then acidified with 6 N HCl. Products 7a and 7b were filtered by suction and recrystallized from ethanol.
2-{3-Methyl-5-[4-(methylthio)benzyl]-6-oxopyridazin-1(6H)-yl}acetic acid (7a)
Mp = 87–89°C; crystallization solvent = EtOH; yield = 99.9%.1H NMR (CDCl
3), d 2.25 (3H, s), 2.51
(3H, s), 3.87 (2H, s), 4.91 (2H, s), 6.69 (1H, s), 7.16 (2H, d, J = 7.6 Hz), 7.25 (2H, d, J = 7.6 Hz).
General procedure for compounds (1) [Cilibrizzi et al., 2009] and (8a-c)
To a cooled (-5°C) and stirred solution of the appropriate carboxylic acid 7a,b (0.90 mmol), in anhy-drous tetrahydrofuran (6 ml), Et3N (3.15 mmol) was
added. After 30 min, the mixture was allowed to warm up to 0°C, and ethyl chloroformate (0.99 mmol) was added. After 1 h, the appropriately substituted ary-lamine (1.80 mmol) was added, and the reaction was
carried out at room temperature (rt) for 12 h. The mixture was then concentrated in vacuo, diluted with cold water (10–15 ml) and extracted with CH2Cl2
(3 ¥ 15 ml). The solvent was evaporated to obtain final compounds 1 and 8a-c, which were purified by column chromatography using cyclohexane/ethyl acetate 1:1 for compounds 1 and 8a,c and cyclohexane/ethyl acetate 2:1 for 8b as eluents.
N-(4-Bromophenyl)-2-{3-methyl-5-[4-(methylthio)
benzyl]-6-oxo-pyridazin-1(6H)-yl}acetamide (8a) Mp = 97–99°C; purified by column chromatogra-phy (cyclohexane/ethyl acetate 1:1); yield = 10%. 1H
NMR (CDCl3) d 2.30 (3H, s), 2.50 (3H, s), 3.89 (2H, s),
4.94 (2H, s), 6.80 (1H, s), 7.17 (2H, d, J = 8.2 Hz), 7.24 (2H, d, J = 8.2 Hz), 7.39 (4H, dd, J = 2.6, 5.7 Hz), 9.01 (1H, exch br s). MS (ESI) Calcd. for C21H20BrN3O2S,
458.37. Found: m/z 458.17 [M + H]+. Anal. Calcd for
C21H20BrN3O2S: C, 55.03; H, 4.40; N, 9.17. Found: C,
54.92; H, 4.39; N, 9.20.
N-(4-Iodophenyl)-2-{3-methyl-5-[4-(methylthio)
benzyl]-6-oxo-pyridazin-1(6H)-yl}acetamide (8b) Mp = 68–70°C; purified by column chromatogra-phy (cyclohexane/ethyl acetate 2:1); yield = 46%. 1H
NMR (CDCl3) d 2.30 (3H, s), 2.51 (3H, s), 3.89 (2H, s),
4.93 (2H, s), 6.80 (1H, s), 7.18 (2H, s, J = 8.4 Hz), 7.24-7.28 (4H, m), 7.61 (2H, d, J = 8.8 Hz), 8.92 (1H, exch br s). MS (ESI) Calcd. for C21H20IN3O2S, 505.37. Found:
m/z 506.11 [M + H]+. Anal. Calcd for C
21H20IN3O2S:
C, 49.91; H, 3.99; N, 8.31. Found: C, 49.86; H, 4.01; N, 8.34.
2-[5-(3-Methoxybenzyl)-3-methyl-6-oxopyridazin-1 (6H)-yl]-N-[4-(methylthio)phenyl]acetamide (8c)
Mp = 166–167°C; purified by column chromatog-raphy (cyclohexane/ethyl acetate 1:1); yield = 99.9%.1H
NMR (CDCl3), d 2.28 (3H, s), 2.45 (3H, s), 3.80 (3H, s),
3.89 (2H, s), 4.95 (2H, s), 6.79–6.85 (4H, m), 7.12– 7.15 (1H, m), 7.27 (1H, t, J = 7.8 Hz), 7.39 (2H, d,
J = 5.0 Hz), 9.13 (1H, exch br s). MS (ESI) Calcd. for
C22H23N3O3S, 409.50. Found: m/z 410.11 [M + H]+.
Anal. Calcd for C22H23N3O3S: C, 64.53; H, 5.66; N,
10.26. Found: C, 64.66; H, 5.64; N, 10.22.
N-(4-Bromophenyl)-2-[5-(3-methoxybenzyl)- 3-methyl-6-oxo-pyridazin-1(6H)-yl]ethanethioamide (9)
Compound 9 was obtained by slow addition of Lawesson’s reagent (0.28 mmol) to a stirred solution of
compound 1 (0.14 mmol) in toluene (3 ml), and the reaction was carried out at reflux for 3 h. The solvent was removed in vacuo, and the mixture was diluted with ice-cold water and extracted with CH2Cl2 (3 ¥ 10 ml).
The crude product was finally purified by flash column chromatography using cyclohexane/ethyl acetate 1:1 as eluent, to yield 9 as an amorphous solid. Mp = 68– 70°C; crystallization solvent = EtOH; yield = 30%. 1H
NMR (CDCl3), d 2.32 (3H, s), 3.81 (3H, s), 3.92 (2H, s),
5.37 (2H, s), 6.81 (1H, s), 6.83-6.87 (3H, m), 7.28 (1H, t, J = 5.0 Hz), 7.48 (2H, d, J = 8.8 Hz), 7.74 (2H, d,
J = 8.8 Hz), 11.46 (1H, exch br s). MS (ESI) Calcd. for
C21H20BrN3O2S, 458.37. Found: m/z 458.17 [M + H]+,
482.36 [M + Na]+, 378.45 [M – Br]+, 231.17 [M –
C8H7BrNS]+. Anal. Calcd for C21H20BrN3O2S: C, 55.03;
H, 4.40; N, 9.17. Found: C, 55.08; H, 4.39; N, 9.19. 4-(4-Methoxybenzyl)-6-methylpyridazine-3(2H)-thione (10)
Lawesson’s reagent (0.87 mmol) was slowly added to a stirred solution of intermediate 5c (0.87 mmol) in toluene (3 ml), and the reaction was carried out at reflux for 2 h. The mixture was cooled and after 1 h stirring in ice-bath, the precipitate was filtered and purified by recrystallization from ethanol. Mp = 191–193°C; crystallization solvent = EtOH; yield = 47%. 1H NMR
(CDCl3), d 2.30 (3H, s), 3.85 (3H, s), 4.11 (2H, s), 6.57
(1H, s), 6.93 (2H, d, J = 8.5 Hz), 7.17 (2H, d,
J = 8.5 Hz), 12.12 (1H, exch br s).
N-(4-Bromophenyl)-2-[4-(4-methoxybenzyl)-6-methylpyridazin-3-ylthio]acetamide (11)
A mixture of compound 10 (0.41 mmol), K2CO3
(0.82 mmol), and N-(4-bromophenyl)-2-chloro aceta-mide (0.61 mmol) in CH3CN (4 ml) was refluxed under
stirring for 1.5 h. After cooling, the solvent was evapo-rated, and the mixture was diluted with cold water. The precipitate was filtered and purified by flash chroma-tography using cyclohexane/ethyl acetate 1:2 as eluent. Mp = 116–118°C; purified by column chromatography (cyclohexane/ethyl acetate 1:2); yield = 96%.1H NMR
(CDCl3), d 2.62 (3H, s), 3.84 (3H, s), 3.87 (2H, s), 4.08
(2H, s), 6.84 (1H, s), 6.92 (2H, d, J = 8.5 Hz), 7.11 (2H, d, J = 8.5 Hz), 7.38 (2H, d, J = 8.8 Hz), 7.48 (2H, d,
J = 8.8 Hz), 10.26 (1H, exch br s). MS (ESI) Calcd. for
C21H20BrN3O2S, 458.37. Found: m/z 458.11 [M + H]+.
Anal. Calcd for C21H20BrN3O2S: C, 55.03; H, 4.40; N,
9.17. Found: C, 54.81; H, 4.38; N, 9.19. 3-Chloro-4-(4-methoxyphenoxy)-6-phenylpyridazine (13)
A suspension of 3-chloro-6-phenylpyridazin-4-ol 12 (1.31 mmol), 4-methoxyphenylboronic acid
(2.62 mmol), copper acetate (1.96 mmol), and Et3N
(2.62 mmol) in CH2Cl2(10 ml) was stirred for 12 h at rt.
The copper salts were filtered off, and the organic layer was extracted with 50% aqueous ammonia (3 ¥ 10 ml), washed with water (10 ml), and dried over Na2SO4. After removal of the solvent in vacuo, the
residue was purified by flash column chromato-graphy using cyclohexane/ethyl acetate 1:1 as eluent. Mp = 124–126°C; purified by column chromatography (cyclohexane/ethyl acetate 1:1); yield = 48%. 1H-NMR
(CDCl3), d: 3.86 (3H, s), 6.99 (2H, d, J = 9.2 Hz),
7.45-7.47 (3H, m), 7.61 (2H, d, J = 9.2 Hz), 7.78-7.80 (2H, m), 7.93 (s, 1H).
4-(4-Methoxyphenoxy)-6-phenylpyridazin-3-amine (14)
A solution of compound 13 in saturated ethanolic ammonia was heated in a sealed stainless steel tube at 180°C for 7 h. The mixture was then concen-trated in vacuo, and the residue was treated with cold diethyl ether to obtain a crude solid that was recovered by suction and recrystallized by ethanol. Mp = 160– 162°C; crystallization solvent = EtOH; yield = 99.9%.
1H-NMR (DMSO), d: 3.79 (3H, s), 6.66 (2H, exch br s),
6.78 (1H, s), 7.01 (2H, d, J = 8.4 Hz), 7.38-7.43 (3H, m), 7.53 (2H, d, J = 8.4 Hz), 7.73 (2H, d, J = 7.2 Hz). 1-(4-Bromophenyl)-3-[4-(4-methoxyphenoxy)-6-phenylpyridazin-3-yl]urea (15a)
To a stirred solution of compound 14 (0.23 mmol) in anhydrous toluene (3 ml), 4-bromophenyl isocyanate (0.26 mmol) was added. The mixture was refluxed for 6 h. After cooling, the solvent was removed under reduced pressure, and the residue was dissolved in CH2Cl2and washed, in turn, with 2 N HCl (3 ¥ 15 ml),
2 N NaOH (3 ¥ 15 ml), and with H2O (15 ml). The
organic layer was dried over Na2SO4and evaporated in
vacuo to obtain the crude products, which were purified
by flash column chromatography using CH2Cl2/CH3OH
(gradient 100:0 to 98:2) as eluent. Mp = 267–269°C; purified by column chromatography (CH2Cl2/CH3OH,
gradient 100:0 to 98:2); yield = 25%.1H-NMR (CDCl 3),
d: 3.65 (3H, s), 6.82 (4H, d, J = 8.8 Hz), 7.42–7.48 (5H, m), 7.64 (2H, d, J = 8.8 Hz), 7.89–7.92 (2H, m), 8.55 (1H, exch br s), 8.80 (1H, s), 9.42 (1H, exch br s). MS (ESI) Calcd. for C24H19BrN4O3, 491.34. Found: m/z
491.11 [M + H]+. Anal. Calcd for C
24H19BrN4O3: C,
58.67; H, 3.90; N, 11.40. Found: C, 58.55; H, 3.89; N, 11.35.
4-Bromo-N-[4-(4-methoxyphenoxy)-6-phenylpyridazin-3-yl]benzamide (15b)
Et3N (0.025 ml) and 4-bromobenzoyl chloride
solution of 14 (0.33 mmol) in anhydrous CH2Cl2(4 ml),
and the reaction was mantained under stirring at 0°C for 3 h. Additional 4-bromobenzoyl chloride (0.67 mmol) was added, and the mixture was stirred at rt for 24 h. The solid residue was removed by filtration, and the organic layer was washed with 6 N NaOH (3 ¥ 10 ml), 6 N HCl (3 ¥ 10 ml) and, finally, with cold water (2 ¥ 10 ml). Drying with Na2SO4and evaporation
of the solvent in vacuo resulted in crude compound 15b, which was purified by flash chromatography using CH2Cl2 as eluent. Mp = 203–204°C; purified
by column chromatography (CH2Cl2); yield = 63%. 1H-NMR (CDCl
3), d: 3.87 (3H, s), 7.02 (2H, d,
J = 8.4 Hz), 7.44–7.49 (3H, m), 7.66 (4H, t, J = 8.4 Hz),
7.83 (2H, s. J = 8.4 Hz), 7.91 (2H, dd, J = 6.0, 2.0 Hz), 8.84 (1H, s), 9.52 (1H, exch br s). MS (ESI) Calcd. for C24H18BrN3O3, 476.32. Found: m/z 476.11 [M + H]+.
Anal. Calcd for C24H18BrN3O3: C, 60.52; H, 3.81; N,
8.82. Found: C, 60.46; H, 3.82; N, 8.85.
Ethyl 2-[4-(4-methoxyphenoxy)-6-phenylpyridazin-3-ylamino]acetate (16)
A mixture of the intermediate 14 (0.33 mmol), K2CO3 (0.66 mmol), and ethyl bromoacetate (1.00
mmol) in CH3CN (4 ml) was refluxed with stirring for
8 h. The mixture was then concentrated, diluted with cold water, and extracted with CH2Cl2(3 ¥ 15 ml). The
organic layer was dried over Na2SO4 and evaporated
in vacuo to obtain the crude product 16, which was
purified by flash chromatography using cyclohexane/ ethyl acetate 2:1 as eluent. Mp = 130–132°C; purified by column chromatography (cyclohexane/ethyl acetate 2:1); yield = 79%. 1H NMR (CDCl 3), d: 1.32 (3H, t, J = 7.2 Hz), 3.83 (3H, s), 4.04 (2H, d, J = 5.2 Hz), 4.26 (2H, q, J = 7.2 Hz), 6.39 (1H, s), 6.98 (2H, d, J = 8.8 Hz), 7.39-7.43 (3H, m), 7.62 (2H, d, J = 8.8 Hz), 7.78 (2H, d, J = 7.6 Hz). 2-[4-(4-Methoxyphenoxy)-6-phenylpyridazin-3-ylamino]acetic acid (17)
A suspension of compound 16 (0.30 mmol) in 6 N NaOH (4 ml) was stirred at rt to 80°C for 2 h. The mixture was first diluted with ice-cold water, acidified with 6 N HCl, and then extracted with CH2Cl2. The
organic layer was dried over Na2SO4and evaporated in
vacuo to obtain the crude product 17, which was
puri-fied by recrystallization from ethanol. Mp = 203–205°C; crystallization solvent = EtOH; yield = 54%. 1H NMR
(CDCl3), d: 3.83 (3H, s), 3.95 (2H, s), 6.42 (1H, s),
6.94–6.97 (2H, m), 7.40–7.48 (3H, m), 7.57 (2H, d,
J = 8.8 Hz), 7.76–7.79 (2H, m).
N-(4-Bromophenyl)-2-[4-(4-methoxyphenoxy)-6-phenylpyridazin-3-ylamino]acetamide (18)
To a cooled (-5°C) and stirred solution of the carboxylic acid 17 (0.14 mmol) in anhydrous tetrahy-drofuran (5 ml), Et3N (0.49 mmol) was added. After
30 min, the mixture was allowed to warm up to 0°C, and ethyl chloroformate (0.15 mmol) was added. After 1 h, 4-bromoaniline (0.28 mmol) was added. The reaction was carried out at room temperature for 5 h. The mixture was then concentrated in vacuo, diluted with cold water (10–15 ml), and extracted with CH2Cl2
(3 ¥ 15 ml). The solvent was evaporated to obtain final compound 18, which was purified by column chroma-tography using cyclohexane/ethyl acetate 1:1 as eluent. Mp = 238–239°C; purified by column chromatography (cyclohexane/ethyl acetate 1:1); yield = 28%. 1H-NMR
(CDCl3), d: 3.83 (3H, s), 4.09 (2H, s), 6.60 (1H, s),
6.96 (2H, d, J = 9.2 Hz), 7.41-7.49 (7H, m), 7.58 (2H, d, J = 9.2 Hz), 7.76 (2H, dd, J = 5.2, 2.8 Hz), 8.25 (1H, exch br s). MS (ESI) Calcd. for C25H21BrN4O3,
505.36. Found: m/z 505.10 [M + H]+. Anal. Calcd for
C25H21BrN4O3: C, 59.42; H, 4.19; N, 11.09. Found: C,
59.53; H, 4.17; N, 11.11.
Pharmacology Cell culture
Human promyelocytic leukemia HL-60 cells stably transfected with FPR1 (HL-60-FPR1), FPR2 (HL-60-FPR2), or FPR3 (HL-60-FPR3) were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, 10 mM HEPES, 100 mg/ml streptomycin, 100 U/ml penicillin, and G418 (1 mg/ml), as previously described [Christophe et al., 2002]. Wild-type HL-60 cells were cultured under the same condi-tions but without G418.
Isolation of human neutrophils
Blood was collected from healthy donors in accor-dance with a protocol approved by the Institutional Review Board at Montana State University. Neutro-phils were purified from the blood using dextran sedimentation, followed by Histopaque 1077 (Sigma Aldrich, St. Louis, MO USA) gradient separation and hypotonic lysis of red blood cells, as previously described [Schepetkin et al., 2007]. Isolated neutro-phils were washed twice and resuspended in HBSS without Ca2+ and Mg2+ (Hank’s balanced salt solution
[HBSS-]; Mediatech, Inc., Herndon, VA). Neutrophil
preparations were routinely>95% pure, as determined by light microscopy, and>98% viable, as determined by trypan blue exclusion.
Ca2+mobilization assay
Changes in intracellular Ca2+were measured with
a FlexStation II scanning fluorometer using a FLIPR 3 calcium assay kit (Molecular Devices, Sunnyvale, CA) for human neutrophils and HL-60 cells. All active com-pounds were evaluated in parent (wild-type) HL-60 cells to verify that agonists are inactive in nontrans-fected cells (data not shown). Human neutrophils or HL-60 cells, suspended in HBSS- containing 10 mM
HEPES, were loaded with Fluo-4 AM dye (Invitrogen, Eugene, OR) (1.25 mg/ml final concentration) and incu-bated for 30 min in the dark at 37°C. After dye loading, the cells were washed with HBSS- containing 10 mM
HEPES, resuspended in HBSS containing 10 mM HEPES and Ca2+ and Mg2+ (HBSS+), and aliquotted
into the wells of a flat-bottomed, half-area-well black microtiter plates (2 ¥ 105 cells/well). The compound
source plate contained dilutions of test compounds in HBSS+. Changes in fluorescence were monitored
(lex= 485 nm, lem= 538 nm) every 5 s for 240 s at room
temperature after automated addition of compounds. Maximum change in fluorescence, expressed in arbi-trary units over baseline, was used to determine agonist response. Responses were normalized to the response induced by 5 nM fMLF (Sigma Chemical Co., St. Louis, MO) for HL-60-FPR1 and neutrophils, or 5 nM Trp-Lys-Tyr-Met-Val-D-Met-NH2 (WKYMVm)
(Cal-biochem, San Diego, CA) for FPR2 and HL-60-FPR3 cells, which were assigned a value of 100%. Curve fitting (5–6 points) and calculation of EC50were
performed by nonlinear regression analysis of the dose– response curves generated using Prism 5 (GraphPad Software, Inc., San Diego, CA).
Chemotaxis assay
Neutrophils were suspended in HBSS+containing
2% (v/v) fetal bovine serum (FBS) (2 ¥ 106 cells/ml), and chemotaxis was analyzed in 96-well ChemoTx chemotaxis chambers (Neuroprobe, Gaithersburg, MD), as previously described [Schepetkin et al., 2007]. In brief, lower wells were loaded with 30 ml of HBSS+
containing 2% (v/v) FBS and the indicated concentra-tions of test compound, DMSO (negative control), and 1 nM fMLE as a positive control. The number of migrated cells was determined by measuring ATP in lysates of transmigrated cells using a luminescence-based assay (CellTiter-Glo; Promega, Madison, WI), and luminescence measurements were converted to absolute cell numbers by comparison of the values with standard curves obtained with known numbers of neutrophils. The results are expressed as percentage of negative control and were calculated as follows: (number of cells migrating in response to test
compounds/spontaneous cell migration in response to control medium) ¥ 100. EC50 values were determined
by nonlinear regression analysis of the dose–response curves generated using Prism 5 software.
Molecular modeling
The FPR1 homology model was created using the crystal structure of bovine rhodopsin, as reported pre-viously [Movitz et al., 2010]. A Protein Data Bank (PDB) file of the homology model was loaded into the Molegro Virtual Docker (MVD) program (MVD 2010.4.2, Molegro ApS, Katrinebjerg, Denmark). The position of the ligand binding site in the model was recently localized by docking studies of different FPR1 agonists [Movitz et al., 2010; Khlebnikov et al., 2012]. Docking search space was defined as a sphere centered at the carbonyl carbon of the Ala residue in the FPR1 peptide agonist Ac-QAWF in its docking pose obtained by Movitz et al. [2010]. The radius of the sphere was adopted to be equal to 11 Å. This search space encom-passed the whole Ac-QAWF molecule and included, at least partially, 36 residues of FPR1. Among these, side chains of 23 residues closest to the center of the search space were set flexible during the docking simulation.
For FPR2 homology modeling, the primary amino acid sequence of FPR2 was submitted to the Phyre2 (Protein Homology/analogy Recognition Engine V2.0) protein fold recognition server (Structural Bioinformat-ics Group, Imperial College, London; http://www.sbg. bio.ic.ac.uk/phyre2) [Kelley and Sternberg, 2009], and the homology model, created using the crystal structure of bovine rhodopsin, was optimized, as described recently [Schepetkin et al., 2013].
Taking into account a lack of structural informa-tion about any ligand-receptor complex with FPR2, we tried to locate cavities in the macromolecule obtained by homology modeling in order to identify the search space for docking. Use of the MVD “Detect cavity” module with probe size 1.2 Å gave two cavities with volumes of 241 and 25 Å3in the region of the binding
site. Positions of these two cavities reflect an asymmet-ric dumb-bell shape of the binding site. Hence, for FPR2, we also chose a spherical search space with a default radius 15 Å centered at the terminus of the larger cavity directed to the smaller one. This sphere embraced two cavities and eight residues reported by Fujita et al. [Fujita et al., 2011]. Side chains of 45 resi-dues closest to the center of the sphere were considered flexible in the docking study.
Before docking, structures of the compounds were preoptimized using HyperChem software (Hyper-cube, Inc., Gainesville, FL) with MM+ force field and saved in Tripos MOL2 format. The ligand structures
were then imported into the MVD program with the following options enabled: “Create explicit hydrogens,” “Assign charges (calculated by MVD),” and “Detect flexible torsions in ligands.” Selected molecules were docked into FPR1 and FPR2 using the search spaces indicated above with a rigid receptor structure. Ligand flexibility was accounted for with respect to torsion angles autodetected in MVD. MolDock score functions were used with 0.3 Å grid resolution. The “Internal H-bond” option was activated in the “Ligand evalua-tion” menu of Docking Wizard. Fifteen docking runs were performed for each molecule with side chain flex-ibility enabled for the residues mentioned above. The postprocessing options “Energy minimization” and “Optimize H-bonds” were applied after docking.
RESULTS AND DISCUSSION
We synthesized a series of new pyridazin-3(2H)-one-based thioderivatives and pyridazine analogs and screened them in order to identify novel molecules able to activate human neutrophils through FPRs. The final compounds were evaluated for their ability to induce intracellular Ca2+ flux in HL-60 cells transfected with
FPR1, FPR2, or FPR3, as it is possible to estimate FPR binding using this assay system [Prossnitz et al., 1991; Didsbury et al., 1992]. Both EC50 values and relative
efficacy, compared with the peptide agonists (fMLF
and WKYMVm) and previously described pyrida-zinones 1–3 (Fig. 1 and [Cilibrizzi et al., 2009]), were determined. All compounds were also evaluated in wild-type, nontransfected HL-60 cells to verify re-sponse specificity. Moreover, the compounds that showed the best profile were selected to evaluate their chemotactic activity and capacity to mobilize Ca2+ in
human neutrophils.
Analysis of pyridazinone derivatives containing a methylthio group in the phenyl ring at position C-4 (Table 1) showed that substitution of OCH3at this
posi-tion of the previously published FPR2-specific agonist 2 (Fig. 1 and [Cilibrizzi et al., 2009]) with SCH3resulted
in compound 8a, which was inactive for all FPRs. Thus, differences in steric hindrance between oxygen and sulfur appear to result in loss of agonist activity. Sur-prisingly, the iodine analog, compound 8b, had agonist activity in the micromolar range for both FPR1 and FPR2. Similarly, substitution of OCH3in the para
posi-tion of the phenylacetamide linker of reference com-pound 3 (Fig. 1 and [Cilibrizzi et al., 2009]) with SCH3
(compound 8c) did not change FPR agonist activity, keeping the potency at micromolar levels. These data suggest that it is possible to modify the para position of phenylacetamide side chain, resulting in compounds with similar activity. In contrast, transformation of our lead compound 1 (Fig. 1 and [Cilibrizzi et al., 2009]) by substitution of the oxygen atom in the amide bridge
TABLE 1. Functional Activity of New Pyridazin-3(2H)-One Thioderivatives 8a-c and 9 in HL-60 Cells Expressing Human FPR1, FPR2, or FPR3, in Comparison with Reference Compounds 1–3*
N N X NH R1 O R Compd. R X R1 Ca2+mobilization EC 50(mM) and efficacy (%)† FPR1 FPR2 FPR3 1* OCH3(m) O Br 3.4⫾ 1.6 (75) 3.8⫾ 1.5 (70) N.A.
9 OCH3(m) S Br N.A. N.A. N.A.
2* OCH3(p) O Br N.A. 2.4⫾ 0.9 (70) N.A.
8a SCH3(p) O Br N.A. N.A. N.A.
3* OCH3(m) O OCH3 7.7⫾ 2.5 (65) 14.4⫾ 2.0 (35) N.A.
8c OCH3(m) O SCH3 2.2⫾ 0.69 (70) 8.2⫾ 2.5 (40) N.A.
8b SCH3(p) O I 2.3⫾ 0.72 (80) 9.4⫾ 2.7 (60) N.A.
fMLF 0.01 20.4 1.9
WKYMVm 0.5 0.001 0.01
*Values of activity and efficacy are from Cilibrizzi et al. [2009].
†N.A., no activity was observed (no response was observed during first 2 min after addition of compounds under investigation) considering the limits of efficacy>20% and EC50<50 mM. EC50values are presented as the mean⫾ standard deviation of three independent experiments, in
which median effective concentration values (EC50) were determined by nonlinear regression analysis of the dose–response curves (5–6 points)
generated using GraphPad Prism 5 with 95% confidential interval (P< 0.05). Efficacy (in parentheses) is expressed as % of the response induced by 5 nM fMLF (FPR1) or 5 nM WKYMVm (FPR2 and FPR3).
with a sulfur atom led to thioamide 9, which was inac-tive. Lastly, none of the pyridazine analogs (11, 15a,b, and 18) exhibited FPR agonist activity.
Both FPR1/FPR2 agonists 8b and 8c activated human neutrophil Ca2+ flux (EC
50= 2.2 and 2.1 mM,
respectively) and chemotaxis (EC50= 2.9 and 2.4 mM,
respectively). Representative dose–response curves for these activities are shown in Figure 4.
Molecular docking of selected thioderivatives into the FPR1 and FPR2 binding sites was performed to assess how the presence of the sulfur atom might influ-ence the binding modes of the compounds as compared with their oxygen-containing analogs. Taking into account that FPR1 has a more open and broader binding site than FPR2, the docking results were ana-lyzed in terms of partial interactions of ligands with FPR1 residues. As shown in Figure 5, compounds 8a, 8b, 1, and 9 occupy different locations within the receptor. Arg205 is one of the key residues contributing to FPR1 agonist activity [Mills et al., 2000; Khlebnikov et al., 2012], and the highly active peptide fMLE and 2-(benzimidazol-2-ylthio)-N-phenylacetamide-derived FPR1 agonists strongly interacted with Agr205, includ-ing formation of H-bonds [Khlebnikov et al., 2012]. Likewise, our docking studies indicated that compound 8b had interaction energy of -20.02 kcal/mol with Arg205 and was H-bonded to this residue, whereas the A
B
Fig. 4. Activation of human neutrophils by selected compounds. (A)
Representative kinetics of neutrophil intracellular Ca2+flux after
treat-ment with compound 8b or fMLF. Human neutrophils were treated with different concentrations of the compound 8b, 5 nM fMLF, or 1% DMSO (negative control), and Ca2+flux was monitored for the
indi-cated times. The data are from one experiment that is representative of three independent experiments. (B) Human neutrophil chemotaxis toward the indicated concentrations of compounds 8b (䊏) and 8c (䊊) was determined, as described under Materials and Methods. The data are presented as the mean⫾ standard deviation of duplicate samples from one experiment that is representative of two indepen-dent experiments.
Fig. 5. Docking poses of selected pyridazine derivatives within the
FPR1 binding site. (A) Poses of active compound 8b (blue) and inactive compound 8a (brown). (B) Poses of active compound 1 (yellow) and inactive compound 9 (sage). [Color figure can be viewed in the online issue which is available at wileyonlinelibrary.com]
inactive derivative 8a had a very weak, nonbonded attraction to Arg205. Additionally, molecule 8b formed H-bonds with Thr265, whereas 8a was not H-bonded to any FPR1 residue (Fig. 5A). A similar situation occurred for the active and inactive compounds 1 and 9, respectively (Fig. 5B). Active pyridazine 1 formed an H-bond with Arg205 and had total interaction energy of 32.9 kcal/mol with this residue. On the other hand, compound 9 had a much weaker, nonbonded interac-tion with Arg205 (-8.82 kcal/mol).
Pairs of sulfur- and oxygen-containing compounds were also compared for their ability to interact with FPR2. As an example, a detailed comparison of docking poses for the active oxygen-containing compound 2 and its inactive methylthio analog 8a showed that com-pound 8a did not have any satisfactory binding mode within the FPR2 binding site and that its best docking pose had significant repulsive interactions with Tyr277 and Ile280 of FPR2. These steric conflicts made it impossible for the sulfur-containing ligand to effectively penetrate into the receptor cavity. In spite of H-bonding with Asn171 and Asp173, this docking pose was energetically unfavorable, and its MolDock score was about 76 kcal/mol higher than that of the active methoxy-derivative 2. In comparison, the best pose of agonist 2 was well incorporated into the cavity of the 241 Å3 binding site obtained with the MVD “Detect
Cavity” feature (see Materials and Methods). The closest nonvalent contact of 2.48 Å occurred between the oxygen atom of the ligand 2 methoxy group and Thr168. An analogous pose would be impossible for compound 8a whose methylthio group is far more bulky than the methoxy substituent in molecule 2.
The docking poses of active oxygen-containing derivatives 1–3 and 8c fit the FPR2 cavity well (Fig. 6A). In contrast, the inactive sulfur-containing pyridazine derivatives 8a and 9 had molecular frag-ments outside the cavity, which caused steric hin-drances and prevented an effective binding of the ligands with FPR2 (Fig. 6B). Such positions of com-pounds 8a and 9 within the binding site were restricted by the bulkiness of sulfur-containing molecular frag-ments. Hence, these molecules cannot adopt more suitable conformations that are possible for the corre-sponding oxygen-containing derivatives.
Overall, our docking studies showed that binding modes of oxygen-containing pyridazine derivatives are quite different from their thio-analogs. In the case of FPR2, this is caused mainly by bulkiness of sulfur-containing groups, i.e. by a higher van der Waals radius and longer valence bonds formed by sulfur atoms with respect to oxygen atoms. It is also known that sulfur forms much weaker H-bonds than oxygen [Wood et al., 2008]. Although the importance of H-bonds for binding
to a receptor was shown here for some active FPR1 and FPR2 agonists, the docking studies indicated that replacement of the sulfur atom by oxygen did not lead to emergence of an H-bond with participation of this oxygen atom in docking. We also confirmed that most active FPR1 agonists strongly interact with Arg205 of the receptor, as described previously [Mills et al., 2000; Khlebnikov et al., 2012].
In conclusion, we synthesized several novel FPR agonists with pyridazinone or pyridazine scaffolds as modified analogs of the previously reported series of FPR agonists [Cilibrizzi et al., 2009]. Among the pyridazinone analogs tested, thioderivatives 8b and 8c were mixed FPR1/FPR2 agonists with micromolar activity, whereas all pyridazine analogs and thioderiva-tives 8a and 9 were completely inactive. Furthermore, Fig. 6. Docking poses of selected pyridazine derivatives within the
FPR2 binding site. The cavity of FPR2 found by the MVD program is shown by a green grid, and residues closest to the cavity are shown. (A) Poses of active compounds 1 (yellow), 2 (light-blue), 3 (green), and
8c (blue) fit the receptor cavity well. (B) Poses of inactive compounds 8a (magenta) and 9 (red) have molecular fragments outside of the
cavity (p-bromophenyl groups of compounds 8a and 9; methyl and methythio groups of compound 8a). [Color figure can be viewed in the online issue which is available at wileyonlinelibrary.com]
molecular docking studies suggested that the inactivity of these thioderivatives was due to a weak H-bonding acceptor and bulkiness of sulfur-containing molecular fragments. These findings confirmed the crucial impor-tance of the pyridazinone scaffold for the activity of this class of compounds.
ACKNOWLEDGMENTS
This work was supported in part by National Insti-tutes of Health grant GM103500 and the Montana State University Agricultural Experimental Station.
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